Recombinant Polypterus ornatipinnis NADH-ubiquinone oxidoreductase chain 3 (MT-ND3)
Description
Introduction to NADH-ubiquinone oxidoreductase chain 3
NADH-ubiquinone oxidoreductase chain 3 (MT-ND3) is one of the core subunits of mitochondrial Complex I, a crucial component of the electron transport chain. Complex I functions as an entry point to the electron transport chain in mitochondria and many aerobic bacteria, coupling NADH oxidation and quinone reduction to proton translocation across the inner mitochondrial membrane . This energy conversion process is fundamental to cellular respiration and ATP production in eukaryotic organisms. The MT-ND3 protein specifically contributes to the hydrophobic domain of Complex I that resides within the inner mitochondrial membrane, facilitating the proton pumping mechanism that drives oxidative phosphorylation.
In the context of Polypterus ornatipinnis, MT-ND3 represents one of the mitochondrially-encoded subunits of Complex I. The gene encoding this protein, MT-ND3, is located in the mitochondrial genome, consistent with the evolutionary pattern observed in most eukaryotes where the hydrophobic subunits of Complex I are encoded by mitochondrial DNA rather than nuclear DNA . This mitochondrial localization of the gene creates unique challenges for studying the protein, making recombinant expression systems particularly valuable for obtaining sufficient quantities for research purposes.
Biological Significance
The MT-ND3 protein holds significant biological importance as part of respiratory Complex I, which is central to energy transduction in cells. Dysfunctions in Complex I components, including MT-ND3, have been linked to numerous neuromuscular and neurodegenerative diseases, as well as to oxidative stress and aging processes . In humans, variants in the MT-ND3 gene can cause serious conditions such as Leigh syndrome and mitochondrial complex I deficiency, characterized by reduced ATP synthesis and impaired energy metabolism . The study of MT-ND3 from various species, including Polypterus ornatipinnis, provides comparative insights that enhance our understanding of mitochondrial function across different evolutionary lineages.
Function in Mitochondrial Respiration
Recombinant Polypterus ornatipinnis MT-ND3 serves as a crucial component of Complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3), which catalyzes the first step in the mitochondrial electron transport chain . Complex I couples the oxidation of NADH to the reduction of ubiquinone, transferring electrons through a series of iron-sulfur clusters and other redox centers. This electron transfer process is accompanied by the translocation of protons across the inner mitochondrial membrane, contributing to the generation of the proton motive force that drives ATP synthesis via ATP synthase.
As one of the seven hydrophobic subunits encoded by the mitochondrial genome, MT-ND3 participates in the membrane-embedded domain of Complex I . This domain is responsible for proton translocation, making MT-ND3 directly involved in the energy conversion process. The exact mechanism by which MT-ND3 contributes to proton pumping remains an active area of research, but its conservation across species underscores its essential role in this fundamental biological process.
Impact of Mutations
Research on MT-ND3 variants in humans has demonstrated that mutations in this gene can significantly impair Complex I function. For instance, the m.10197G>C variant has been shown to lower MT-ND3 protein levels, causing deficiencies in Complex I assembly and activity, and consequently reducing ATP synthesis . These findings highlight the critical role of MT-ND3 in maintaining proper mitochondrial function and energy production. Similar studies using recombinant Polypterus ornatipinnis MT-ND3 could provide valuable comparative data on the effects of specific amino acid changes on protein function.
Production and Recombinant Expression
The recombinant production of Polypterus ornatipinnis MT-ND3 involves expressing the protein in heterologous systems, typically E. coli, to obtain sufficient quantities for research purposes . This approach overcomes the challenges associated with purifying native MT-ND3 from mitochondrial membranes. The recombinant protein is commonly produced with affinity tags, such as His-tags, to facilitate purification through affinity chromatography techniques.
Expression Systems and Purification
The recombinant Polypterus ornatipinnis MT-ND3 protein is typically expressed in E. coli expression systems . The coding sequence is optimized for bacterial expression and may include modifications to enhance protein solubility and yield. Following expression, the protein is purified to a high level of purity, often exceeding 90% as determined by SDS-PAGE analysis . The purified protein is then formulated in appropriate buffer systems to maintain stability and functionality.
Table 1: Typical Production Parameters for Recombinant Polypterus ornatipinnis MT-ND3
| Parameter | Specification |
|---|---|
| Expression System | E. coli |
| Protein Length | Full Length (1-115 amino acids) |
| Affinity Tag | His-tag (N-terminal) |
| Purity | >90% (SDS-PAGE) |
| Form | Lyophilized powder |
| Storage Buffer | Tris/PBS-based buffer, 6% Trehalose, pH 8.0 |
Codon Optimization Strategies
Recent advances in the field have demonstrated the effectiveness of codon optimization techniques for expressing mitochondrial genes in nuclear expression systems . Although this approach has been primarily applied to human MT-ND3 for therapeutic purposes, similar strategies could be adapted for the expression of Polypterus ornatipinnis MT-ND3. Codon optimization enhances translation efficiency in the heterologous host, potentially increasing protein yield and quality.
Applications in Research
Recombinant Polypterus ornatipinnis MT-ND3 serves as a valuable tool in various research applications, particularly in studies focusing on mitochondrial function, comparative biochemistry, and evolutionary biology. The availability of purified recombinant protein enables detailed biochemical and structural analyses that would be challenging with native protein from biological samples.
Comparative Mitochondrial Research
Polypterus ornatipinnis (Ornate bichir) represents an interesting model for evolutionary studies, as bichirs are considered primitive ray-finned fishes with characteristics of both ray-finned and lobe-finned fishes. Comparative studies of MT-ND3 across different species can provide insights into the evolution of mitochondrial function and the conservation of essential respiratory mechanisms. The recombinant protein allows for direct comparison of biochemical properties with MT-ND3 from other species, potentially revealing adaptations specific to the unique ecological niche of the Ornate bichir.
Reconstitution Protocol
For optimal results, the lyophilized protein should be briefly centrifuged before opening to bring the contents to the bottom of the vial. Reconstitution should be performed using deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL . The addition of glycerol to a final concentration of 5-50% is recommended for long-term storage, with 50% glycerol being the standard formulation . Proper mixing after reconstitution ensures homogeneous protein distribution.
Table 2: Recommended Storage and Handling Parameters
| Parameter | Recommendation |
|---|---|
| Long-term Storage | -20°C or -80°C |
| Working Storage | 4°C (up to one week) |
| Reconstitution Medium | Deionized sterile water |
| Recommended Concentration | 0.1-1.0 mg/mL |
| Glycerol for Storage | 5-50% (final concentration) |
| Avoid | Repeated freeze-thaw cycles |
Product Specs
Note: We will prioritize shipping the format currently in stock. However, if you have specific format requirements, please indicate them in your order notes, and we will fulfill your request.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipment, please inform us in advance, as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. Lyophilized form has a shelf life of 12 months at -20°C/-80°C.
Target Background
Q&A
What is NADH-ubiquinone oxidoreductase chain 3 (MT-ND3) and what is its significance in mitochondrial function?
MT-ND3 is a protein subunit of mitochondrial Complex I (NADH:ubiquinone oxidoreductase), encoded by the mitochondrial genome. It functions as a critical component of the electron transport chain, facilitating the transfer of electrons from NADH to ubiquinone while pumping protons across the inner mitochondrial membrane to establish the electrochemical gradient necessary for ATP synthesis. In Polypterus ornatipinnis (Ornate bichir), MT-ND3 consists of 115 amino acids and is encoded on the mitochondrial H-strand .
The functional significance of MT-ND3 extends beyond basic energy metabolism. Research approaches examining this protein should incorporate membrane potential measurements, oxygen consumption rates, and ROS generation assays to fully characterize its role in cellular bioenergetics. Mutations in MT-ND3 have been linked to various mitochondrial disorders across species, making it an important target for comparative functional studies.
How is the MT-ND3 gene organized within the mitochondrial genome of Polypterus ornatipinnis?
The complete mitochondrial genome sequencing of Polypterus ornatipinnis reveals that MT-ND3 follows the vertebrate consensus mitochondrial gene order . The gene arrangement and the major noncoding region in bichir mtDNA are identical to the established vertebrate consensus, indicating the early establishment of this particular organization in vertebrate evolution .
Within the mitochondrial genome, MT-ND3 is positioned between tRNA genes, consistent with the arrangement seen in other vertebrates. There are several notable features of gene organization:
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Gene overlap patterns similar to other vertebrates, including overlap between ND4 and ND4L
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MT-ND3 is encoded on the H-strand of the mitochondrial genome
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The gene maintains its position in the highly conserved vertebrate mitochondrial gene order
This conservation across evolutionary time underscores the functional importance of not just the gene itself, but its specific positioning within the mitochondrial genome .
What are the optimal storage conditions for recombinant MT-ND3 protein?
For recombinant Polypterus ornatipinnis MT-ND3 protein, optimal storage conditions are critical to maintaining structural integrity and functional activity. Based on established protocols for similar proteins, the following recommendations apply:
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Short-term storage: Store working aliquots at 4°C for up to one week
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Medium-term storage: -20°C in Tris-based buffer with 50% glycerol
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Long-term storage: -80°C in Tris-based buffer with 50% glycerol
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Avoid repeated freeze-thaw cycles as they significantly decrease protein activity
For reconstitution of lyophilized protein, it is recommended to use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL. Adding glycerol to a final concentration of 50% before aliquoting provides protection during freezing . When preparing aliquots, volumes should be sized appropriately for single-use applications to prevent the need for repeated freeze-thaw cycles.
What expression systems are most effective for producing functional recombinant MT-ND3?
While prokaryotic systems like E. coli are commonly used for recombinant protein production due to their simplicity and high yield, membrane proteins like MT-ND3 present specific challenges that may require alternative approaches. Based on available data for similar proteins:
For functional studies of MT-ND3, a balanced approach might involve initial screening in E. coli (as used for Baiomys taylori MT-ND3 ) followed by validation in eukaryotic systems. Critical factors for successful expression include:
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Codon optimization for the host organism
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Incorporation of solubility-enhancing tags (e.g., His tag as used in available recombinant preparations )
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Co-expression with chaperones to promote proper folding
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Careful selection of detergents for membrane protein extraction
How can researchers overcome the challenges in purifying functional MT-ND3?
Purification of membrane proteins like MT-ND3 requires specialized approaches to maintain native structure and function. A systematic purification strategy should include:
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Gentle solubilization using appropriate detergents (DDM, LMNG, or digitonin have proven effective for Complex I components)
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Affinity chromatography utilizing tags (His-tag purification as used for available recombinant MT-ND3 )
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Size exclusion chromatography to remove aggregates
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Ion exchange chromatography for higher purity
Key challenges include maintaining the protein in its native conformation while removing detergent micelles, preventing aggregation, and preserving functional activity. For MT-ND3, which interacts with multiple other subunits in Complex I, maintaining these interaction interfaces is crucial for functional studies.
A methodological advancement is the use of nanodiscs or amphipols to stabilize purified MT-ND3 in a more native-like membrane environment, which has shown superior results for functional characterization compared to traditional detergent micelles.
What experimental approaches are most effective for studying the structure-function relationship of recombinant MT-ND3?
Understanding the structure-function relationship of MT-ND3 requires a multi-faceted approach:
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Cryo-EM Analysis: Given the challenges of crystallizing membrane proteins, cryo-EM has become the method of choice for structural determination of Complex I components, including MT-ND3.
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Cross-linking Mass Spectrometry: This technique can identify interaction partners of MT-ND3 within the complex, providing insights into its integration into Complex I.
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Site-Directed Mutagenesis: Systematic mutation of conserved residues can reveal functional domains. Based on the amino acid sequence (MNLILMMILISSLISTILAIVAFWLPQMNPDMEKLSPYECGFDPLGSARLPFSMRFFLVAILFLLFDLEIALLLPLPWSTHLLDPTLMLMWAFTIIILLTLGLIYEWLQGGLEWAE for Polypterus ornatipinnis MT-ND3 ), researchers should focus on:
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Conserved charged residues which may participate in proton pumping
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Hydrophobic residues that may form transmembrane domains
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Regions conserved across species indicating functional importance
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Activity Assays: NADH:ubiquinone oxidoreductase activity assays coupled with mutations can correlate structural features with function.
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Molecular Dynamics Simulations: In silico approaches can predict conformational changes and protein dynamics in response to various conditions.
Data from these complementary approaches should be integrated to develop a comprehensive model of MT-ND3 function within Complex I.
How can isotope labeling of recombinant MT-ND3 facilitate structural studies?
Isotope labeling is a powerful approach for studying membrane proteins like MT-ND3, particularly when combined with NMR spectroscopy or mass spectrometry. Methodological considerations include:
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Uniform Labeling: Expression in minimal media supplemented with 15N-ammonium sulfate and/or 13C-glucose enables backbone assignment and secondary structure determination.
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Selective Labeling: Incorporating specific labeled amino acids can highlight functional residues. For MT-ND3, selective labeling of conserved residues in the amino acid sequence can provide insights into conformational changes during electron transport.
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Segmental Labeling: For larger proteins, labeling specific segments can reduce spectral complexity.
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Hydrogen-Deuterium Exchange Mass Spectrometry: This technique can identify exposed regions and conformational changes in MT-ND3 during functional cycles.
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Pulse-Chase Experiments: These can reveal assembly kinetics of MT-ND3 into Complex I.
Implementation requires careful optimization of expression systems, as prokaryotic systems like E. coli offer cost-effective labeling but may not provide native folding, while eukaryotic expression systems provide better folding but more challenging and expensive labeling protocols.
How does Polypterus ornatipinnis MT-ND3 compare to MT-ND3 proteins from other vertebrate species?
Comparative analysis of MT-ND3 across vertebrate species provides valuable evolutionary insights. Comparing the amino acid sequences of MT-ND3 from Polypterus ornatipinnis (Ornate bichir) with those from other species reveals:
The amino acid sequence of Baiomys taylori MT-ND3 (MNMIMVISVNIILSSTLILVAFWLPQLNIYTEKANPYECGFDPMSSARLPFSMKFFLVAITFLLFDLEIALLLPIPWAIQMPDMKTMMLTAFILVSILALGLAYEWTQKGLEWTE ) shows both similarities and differences compared to Polypterus ornatipinnis, reflecting their evolutionary distance but functional constraints.
Phylogenetic analysis places bichirs (including Polypterus ornatipinnis) in a unique position as possibly the most primitive living bony fish, making their MT-ND3 sequence particularly valuable for understanding the evolution of this protein .
What can bichir mitochondrial genes tell us about vertebrate evolution?
The complete mitochondrial DNA sequence of Polypterus ornatipinnis provides critical insights into vertebrate evolution:
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The presence of the vertebrate consensus mitochondrial gene order in bichirs documents the early establishment of this particular organization in vertebrate evolution .
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As possibly the most primitive living bony fish (Osteichthyes), the bichir's evolutionary position has been disputed—variously aligned with ray-finned fish (Actinopterygii), lobe-finned fish (Sarcopterygii), or placed in their own group (Brachiopterygii) .
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MT-ND3 and other mitochondrial genes can help resolve these phylogenetic relationships through comparative sequence analysis.
Research approaches should include:
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Maximum likelihood phylogenetic analysis of MT-ND3 across diverse vertebrate species
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Bayesian dating methods to estimate divergence times
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Selective pressure analysis to identify sites under positive or purifying selection
These analyses can help resolve long-standing questions about early vertebrate evolution and provide context for functional studies of MT-ND3.
How can recombinant MT-ND3 be used to study mitochondrial dysfunction?
Recombinant MT-ND3 from Polypterus ornatipinnis serves as a valuable tool for investigating mitochondrial dysfunction through several methodological approaches:
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Reconstitution Studies: Purified recombinant MT-ND3 can be reconstituted with other Complex I components to study assembly defects and their functional consequences.
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Binding Partner Identification: Pulldown assays using tagged recombinant MT-ND3 can identify novel interaction partners within and outside Complex I.
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Competitive Inhibition: Peptides derived from MT-ND3 regions can be used to disrupt specific interactions within Complex I, providing insights into functional domains.
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Antibody Development: Recombinant MT-ND3 can be used to develop specific antibodies for immunoprecipitation or imaging studies of mitochondrial function.
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Cross-Species Functional Complementation: The evolutionary position of bichirs makes their MT-ND3 particularly valuable for complementation studies in model organisms with MT-ND3 dysfunction.
For these applications, researchers should consider using the full-length recombinant protein (115 amino acids for Polypterus ornatipinnis MT-ND3 ) with appropriate tags for detection and purification.
How might MT-ND3 studies contribute to understanding human mitochondrial diseases?
The evolutionary conservation of MT-ND3 makes comparative studies using Polypterus ornatipinnis MT-ND3 relevant to human mitochondrial disease research:
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Mutation Modeling: Known pathogenic human MT-ND3 mutations can be recreated in recombinant Polypterus ornatipinnis MT-ND3 to study functional effects in a controlled system.
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Drug Screening Platforms: Reconstituted systems containing recombinant MT-ND3 can serve as platforms for screening compounds that might rescue function in disease-associated variants.
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Structure-Based Drug Design: Structural information from bichir MT-ND3 can inform the development of small molecules targeting specific domains of human MT-ND3.
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Evolutionary Medicine Insights: The ancestral features of bichir MT-ND3 can provide context for understanding which mutations are likely to be pathogenic in humans based on evolutionary conservation.
This translational research approach bridges the gap between basic evolutionary biology and clinical applications, leveraging the unique evolutionary position of bichirs to gain insights relevant to human health.
